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Abstract

Interactions between macromolecules are the basic events of life. Their characterization is thus a key to
understanding the physiology and the underlying specificity of these events. Not only understanding of the
final states (where the molecules get closest together), also understanding of the course of approach is
mandatory to gain insight into the dynamical picture of molecular processes in a living organism.
We have made an attempt to gain insight into the distance dependence of interactions by investigating
the tight interaction between proteases and their inhibitors. Their interactions have been studied by the
combined use of X-ray crystallography, protein kinetics (enzyme kinetics and surface plasmon resonance)
and atom force microscopy. Atomic force measurements provided the distance between the potential
minimum and the transition state in the Bell's model. Additional information about the distance dependence
was obtained by the kinetics of inactivated cathepsins. These data combined with the structures and models
of complexes of inactivated cathepsins with stefin A, provided insight in the energetics of the distance
dependence. The interactions between the studied proteases and inhibitors are taking place in the range
below 10 Å (8.5 Å was the distance measured by the atom force microscopy). In a crude approximation, the
interaction energy appears to be in a linear relationship with the intermolecular distance. These results shed
light also on understanding of the final binding states, the geometry of which are best revealed by X-ray
crystallography. Several determined structures thus provide insight into the mechanism of binding.
3-dimensional structures of complexes of reduced cathepsin V and stefin A, the MMTS-blocked
cathepsins L and V in complex with stefin A, and the complex of the exopeptidase cathepsin B with stefin
A were determined. They have shown that the modification of the active site cysteine residue prevents the
genuine binding of the inhibitor to the protease, however, the complex is still formed. The binding of the Nterminal
trunk and the first loop of the stefin A to cathepsins is well defined and is very similar between
various cathepsins, whereas the second loop in stefin A exhibits large flexibility. We have also shown that
the occluding loop in the cathepsin B is flexible and can adopt different conformations, depending on the
size and shape of the inhibitor bound to the active site cleft. In contrary to the three loops in stefin A,
clitocypin utilizes only two loops, which bind to the active site cleft. The crystal structures of cathepsin V
in the complex with clitocypin enabled us to explain the previously unknown inhibition mechanism in
atomic detail. The most interesting feature of this interaction is the peptide bond flip, which occurs prior to
or concurrently with the inhibitor docking and enhances the inhibition by the formation of an additional
hydrogen bond.